The edible mushroom Agaricus bisporus (Lange) Imbach var. bisporus, a microorganism belonging to the basidiomycete fungi, is widely cultivated around the world. In Europe and North America, it is the most widely cultivated mushroom species. The value of the annual Agaricus bisporus mushroom crop in the United States was about $1,110,000,000 in 2012-2013, according to the National Agricultural Statistics Service, Agricultural Statistics Board, U.S. Department of Agriculture (Aug. 20, 2013).
Cultures of Agaricus, like those of other microorganisms, are prepared, maintained, propagated and stored on sterile media using microbiological laboratory methods. Sterile tools and aseptic techniques are used within clean rooms or sterile transfer hoods to manipulate cells of the pure cultures for various purposes including clonal propagation and for the development of new strains using diverse techniques including spore germinations on sterile growth media and controlled matings on sterile growth media. Commercial culture inocula including mushroom ‘spawn’ and ‘casing inoculum’ are also prepared using large-scale microbiological production methods, for example by aseptically introducing inoculum of a pure culture of a strain of Agaricus bisporus into from one to 14,000 liters of sterilized growth media under sterile conditions, and are provided to the end user as pure cultures on sterile growth media contained within sterile packaging.
Mushrooms are cultivated commercially within purpose-built structures on dedicated farms. While there are many variations on methods, the following description is typical. Compost prepared from lignocellulosic material such as straw, augmented with nitrogenous material, is finished and pasteurized within a suitable facility. Mushroom spawn, which comprises a sterilized friable ‘carrier substrate’ onto which a pure culture of one mushroom strain has been aseptically incorporated via inoculum and then propagated, is mixed with the pasteurized compost and is incubated for approximately 13 to about 19 days at a controlled temperature, during which time the mycelium of the mushroom culture colonizes the entire mass of compost and begins to digest it. A non-nutritive ‘casing layer’ of material such as peat is then placed over the compost to a depth of from about 1.5 to about 2 inches. Additional ‘casing inoculum’ incorporating the same mushroom culture may be incorporated into the casing layer to accelerate the formation and harvesting of mushrooms, and improve uniformity of the distribution of mycelium and mushrooms in and on the casing surface. Environmental conditions, including temperature and humidity, in the cropping facility are then carefully managed to promote and control the transition of the culture from vegetative to reproductive growth at the casing/air interface. In a further about 13 to about 18 days after casing, mushrooms will have developed to the correct stage for harvest and sale. A flush of mushrooms comprising the original culture will be picked over a 3 to 4 day period. Additional flushes of mushrooms appear at about weekly intervals. Commercially, two or three flushes of mushrooms are produced and harvested before the compost is removed and replaced in the cropping facility.
Seventy to ninety-five percent of the Agaricus mushrooms cultivated in the United States, Europe, and elsewhere have a white pileus color, in accordance with consumer preferences. Market requirements for white mushrooms in the USA and elsewhere are narrow and precise for many observable phenotypic traits such as size, shape, color, color retention, firmness, and related traits such as shelf life. Consequently, genetically different strains of commercially successful white Agaricus bisporus mushrooms are not easily differentiated on the basis of appearance of the mushrooms, which must conform to the relatively strict market requirements. Strains may, to some extent, be differentiated on the basis of traits associated with the mushroom, such as mushroom size, mushroom shape (e.g., cap roundness, flesh thickness), color (i.e., white cap versus brown cap), surface texture (e.g., cap smoothness), tissue density and/or firmness, delayed maturation, basidial spore number greater than two, sporelessness, increased dry matter content, improved shelf life, and reduced bruising, as well as traits associated with the culture itself, and/or products incorporating the culture, and/or crops incorporating the culture, including increased crop yield, altered distribution of yield over time, decreased spawn to pick interval, resistance to infection by, symptoms of, or transmission of bacterial, viral or fungal diseases, insect resistance, nematode resistance, ease of crop management, suitability of crop for mechanical harvesting, and behavioral responses to environmental conditions including stressors, nutrient substrate composition, seasonal influences, farm practices, self/non-self interactions (compatibility or incompatibility) with various mushroom strains, to give some examples. Strains may also be differentiated based on their genotypic fingerprint (presence of specific alleles at defined marker loci in the nuclear or mitochondrial genome). Strains may have different ancestry, which will be reflected directly by the genotype, and indirectly, in some cases, by the phenotype.
Circa 1980, the first two white hybrid strains of A. bisporus, developed by a laboratory at Horst, the Netherlands, were introduced into commercial cultivation. These two “Horst” strains, called U1 and U3, are closely related hybrid strains produced by matings between two pre-existing white cultivated strains, as per M. Imbernon et al., Mycologia, 88(5), 749-761 (1996), herein incorporated by reference. The two parents of U1 and U3 are commercial strains belonging to two longstanding categorical types of strains known as the ‘smooth-white’ (SW) strains and the ‘off-white’ (OW) strains. The original homokaryons (or ‘lines’) obtained from the SW and OW strains, and used in the hybridization that produced the U1 strain, were designated H39 and H97 respectively; these cultures may no longer exist (A. Sonnenberg, pers. comm.). However, a number of laboratories have deheterokaryotized the U1 strain and obtained neohaplont cultures incorporating one or the other nuclear type corresponding to those contributed by H39 or H97, as well as the mitochondrial type of U1. We refer to these two types of neohaplonts of U1 categorically as the SWNC and OWNC lines or homokaryons, respectively. An OWNC line designated ‘H97’ was deposited in the public culture collection of the Fungal Genetics Stock Center of Kansas, USA, by A. Sonnenberg, under the number 10389, and in the public collection of the American Type Culture Collection of Maryland, USA, under the number MYA-4626. The genome of H97 was sequenced and placed in the public domain by the Joint Genome Institute of California, USA (Morin et al. 2012, herein incorporated by reference).
The U1 strain is thought to be the direct progenitor of all other white A. bisporus mushrooms currently cultivated in most regions of the world. Many commercial mushroom strains developed from U1 such as A-15 and S130 meet the criteria for Essentially Derived Varieties (as the term is applied to plant varieties, e.g. in the Plant Variety Protection Act) of U1, having been developed from spores of the initial strain which retain the great majority of the parental genotype (this behavior was shown by R. W. Kerrigan et al. in Genetics, 133, 225-236 (1993), herein incorporated by reference). A group of strains developed either by cloning or by spore culture, or by any other method of ‘essential derivation’ as discussed below, from a single progenitor (as opposed to outbreeding between two different progenitors) is called a derived lineage group. Except for relatively minor acquired genetic differences all white strains developed within the Horst U1 derived lineage group share a single composite N+N heterokaryotic genotype, or a subset of that genotype, with the original U1 strain. For this reason, modern white Agaricus mushroom cultivation is effectively a monoculture.
Agaricus bisporus has a reproductive syndrome known as amphithallism, in which two distinct life cycles operate concurrently. As in other fungi, the reproductive propagule is a spore. Agaricus produces spores meiotically, on a meiosporangium known as a basidium. In a first life cycle, A. bisporus spores each receive a single haploid postmeiotic nucleus; these spores are competent to mate but not competent to produce mushrooms. These haploid spores germinate to produce homokaryotic offspring or lines which can mate with other compatible homokaryons to produce novel hybrid heterokaryons that are competent to produce mushrooms. Heterokaryons generally exhibit much less ability to mate than do homokaryons. This life cycle is called heteromixis, or more commonly, outbreeding. This life cycle operates but typically does not predominate in strains of Agaricus bisporus var. bisporus. 
A second, inbreeding life cycle called intramixis predominates in most strains of Agaricus bisporus var. bisporus. Most spores receive two post-meiotic nuclei, and most such pairs of nuclei consist of Non-Sister Nuclear Pairs (NSNPs) which have a heteroallelic genotype at most or all centromeric-linked loci including the MAT locus. That MAT genotype determines the heterokaryotic phenotype of these offspring, which are reproductively competent and can produce a crop of mushrooms. Unusually among eukaryotes, relatively little chromosomal crossing-over is observed to have occurred in postmeiotic offspring of A. bisporus; empirically, very little heteroallelism (analogous to heterozygosity) is lost among heterokaryotic offspring of a heterokaryotic strain. Consequently, parental and offspring heterokaryotic genotypes and phenotypes tend to closely resemble each other, as noted above; for this reason, essential derivation, e.g., the production of Essentially Derived Varieties (EDVs), is a familiar strain development technique among commercial mushroom spawn producers.
There is a need for commercially acceptable A. bisporus strains with different genotypes, relative to the U1 derived lineage group, for two reasons. First, strains incompatible with strains of the U1 derived lineage group are known to retard the spread of viral diseases between strains, which diseases are known problems in the commercial mushroom industry. Second, it is well understood that when an agricultural crop industry relies extensively on a single genetic lineage (i.e., creates a commercial monoculture as now exists for the white-capped U1 lineage-group of A. bisporus), there is an increased risk of unpredictable, catastrophic crop failure on a facility-wide or industry-wide scale. Therefore from a risk management and food security perspective, it is highly desirable to simultaneously provide both genetic diversification and commercially acceptable performance and crop characteristics.